Enhancing In Vitro Multiplication and Acclimatization of Blackberry (Rubus L.) Through Sterilization Optimizing and Growth Regulator Use

Blackberry (Rubus L.) cultivation in Kazakhstan is constrained by the limited availability of certified planting material and the absence of standardized micropropagation protocols adapted to local conditions. This study aimed to optimize the key stages of in vitro culture for the cultivars ‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’, including explant sterilization, culture initiation, shoot multiplication, and acclimatization. A sequential sterilization scheme using 70% ethanol followed by 1% sodium hypochlorite ensured high explant survival. Shoot initiation was most efficient on MS medium supplemented with 0.1 mg/L BAP, whereas multiplication was enhanced by 0.5 mg/L BAP and 0.1 mg/L GA3. In the subsequent rooting stage, microcuttings formed stable root systems under ex vitro conditions in agroboxes, confirming that the optimized protocol ensured not only high survival during initiation but also a successful transition to the rooting phase, which is essential for further acclimatization. During ex vitro acclimatization, the application of humic acid, nanosilicon, or succinic acid improved survival under agrobox microventilation. The developed approach provides a reliable framework for producing healthy, adapted plants of the evaluated cultivars and contributes to establishing domestic propagation systems for reducing reliance on imported planting material.